Structure-Activity Relationship Studies of Peptide Beta-Lactones as Proteasome Inhibitors and Anticancer Agents ! PROJECT SUMMARY/ABSTRACT The Ubiquitin-proteasome pathway is the principal mechanism by which eukaryotic cells degrade intracellular proteins, and is therefore critical to many cellular processes such as regulation of the cell cycle, immune response and of apoptosis. The inhibition of the proteasome function is a clinically validated strategy for the treatment of multiple myeloma. Current FDA-approved therapies based on the inhibition of the proteasome have transformed the treatment of multiple myeloma and related cancers; however, significant barriers dealing with off-target toxicity, and development of resistance are still to be overcome. New proteasome inhibitors with different binding modes and improved selectivity are critically needed. The cystargolides are two recently isolated beta-lactone natural products with significant proteasome inhibition activity. Our group described the first total synthesis and determined the absolute stereochemistry of the cystargolides. The work also delivered two derivatives that possess ~100-fold enhanced activity as proteasome inhibitors. Moreover, these analogs showed to inhibit MCF-7 breast cancer growth at low micromolar concentration. Since our initial studies, we have also discovered an analogue that is more potent proteasome inhibitor (IC50 = 3.1 nM) than the clinically used drug carfilzomib (about 10 nM in the same assay). Additionally, our preliminary data suggest that our analogues are selective towards the immunoproteasome. This remarkable activity and selectivity places the cystargolide scaffold as an excellent starting point for the development of new proteasome inhibitors with improved anticancer activity. We hypothesize that the cystargolide scaffold can be optimized to deliver proteasome inhibitors that are both highly potent and selective. ! This proposal describes a structure-activity relationship study on the cystargolide scaffold. We will use synthetic and medicinal chemistry strategies to study 1) Structural diversifications of the beta- lactone fragment, 2) Non-pharmacophoric substitutions on the cystargolide scaffold, and utilize 3) biological evaluations of the cystargolide analogs, to identify potent and selective proteasome inhibitors and anticancer agents. The development of novel proteasome inhibitors may also benefit non-cancer applications such as treatment for inflammatory and autoimmune diseases as well as the control of malaria, expanding the potential impact to those fields of public health.